The epidermal growth factor family of transmembrane tyrosine kinase receptors, including EGFR (ErbB1 or HER1), ErbB2 (HER2), ErbB3 (ERBB3 or HER3) and ErbB4 (HER4) are involved in regulating key cellular functions (e.g. cell proliferation, survival, differentiation and migration) through a complex network of intracellular signaling pathways. HER3 differs from the other receptors of this family due to its inactive tyrosine kinase domain, and hence signals via ligand-induced heterodimer formation with other tyrosine kinase receptors (Guy et al, Proc Natl Acad Sci 91: 8132-8136 (1994); Sierke et al, Biochem J 322 (Pt 3): 757-763 (1997)). As a result, the implication of this receptor in tumor progression has long been a mystery. Recently, however, HER3 has gained interest as an allosteric kinase activator of its family members. Especially the heterodimer formed by HER2 and HER3 is said to be an exceptionally strong activator of downstream intracellular signaling (Jura et al, Proc Natl Acad Sci 106: 21608-21613 (2009); Citri et al, Exp Cell Res 284: 54-65 (2003)). This HER2-HER3 signaling pair has even been suggested as an oncogenic unit in HER2-driven breast cancer (Holbro T, et al, Proc Natl Acad Sci 100: 8933-8938 (2003)). In addition, up-regulation of the HER3 receptor has been shown to play an important role for the resistance to tyrosine kinase inhibitors in breast cancers overexpressing HER2 in vitro and in vivo (Sergina et al, Nature 445: 437-441 (2007); Kong et al, PLoS One 3: e2881 (2008); Garrett et al, Proc Natl Acad Sci 108: 5021-5026 (2011)).
However, the importance of HER3 in human cancers is not limited to HER2-driven breast cancers. HER3 has also been shown to be required for tumorigenicity of HER3-overexpressing prostate cancer xenografts in vivo, to maintain in vivo proliferation of a subset of ovarian cancers via an autocrine signaling loop, and to be involved in endocrine resistance of ER+ breast cancer cell lines, to name a few examples (Soler et al, Int J Cancer 125: 2565-2575 (2009); Sheng Q et al, Cancer Cell 17: 412-412 (2010); Liu et al, Int J Cancer 120: 1874-1882 (2007); Frogne et al, Breast Cancer Res Treat 114: 263-275 (2009)). Altogether, these findings demonstrate the potential of the HER3-signaling pathway as an important therapeutic target in human cancers. In addition, HER3 expression has a prognostic value, since high levels of receptor expression are associated with significantly shorter survival time compared with patients that overexpress HER2 (Tanner et al, J Clin Oncol 24(26):4317-23 (2006), Reschke et al, Clin Cancer Res 14(16):5188-97 (2008)).
A relatively large fraction of recently approved therapies directed towards the EGFR and HER2 receptors is based on monoclonal antibodies. In contrast to the well investigated EGFR and HER2 receptor members of the ErbB-family, there are relatively few reports on the use of anti-HER3 antibodies. Ullrich and co-workers have reported that anti-HER3 monoclonal antibodies inhibit HER3 mediated signaling in cell models of breast cancer (van der Horst et al, Int J Cancer 115(4):519-27 (2005)). However, although several successful cancer therapy studies have been reported using full-length monoclonal antibodies, this class of agents is not always optimal for targeting solid tumors (neither for diagnostic nor for therapeutic pay-load purposes). Therapeutic effect is dependent on an efficient distribution of the drug throughout the tumor, and molecular imaging depends on a high ratio between tumor uptake and surrounding normal tissue. Since tumor penetration rate (including extravasation) is negatively associated with the size of the molecule, the relatively large antibody molecule (e.g. IgG) inherently has poor tissue distribution and penetration capacity. Moreover, for molecular imaging, the extraordinarily long in vivo half-life of antibodies results in relatively high blood signals and thereby relatively poor tumor-to-blood contrasts.
Recently, much smaller HER3-specific molecules based on the three-helical bundle scaffold of the Z domain, derived from domain B of Protein A from Staphylococcus aureus, were generated using combinatorial protein engineering (Kronqvist et al, Protein Eng Des Sel 24: 385-396 (2010); WO2011/056124). These Z variants, with subnanomolar affinities for HER3, demonstrated anti-proliferative effects through blockage of ligand-induced HER3-signalling of breast cancer cell lines in vitro (Gostring et al, PLoS One 7:e40023 (2012)). These growth-inhibitory effects were further demonstrated to be a result of competitional HER3 binding between the Z variant molecules and the ligand heregulin.
However, in vivo targeting of the HER3 receptor may be challenging, due to relatively low expression of the receptor on tumor cells. Typical expression levels of 103 to 104 receptors per cell have been reported (Aguilar et al, Oncogene 18: 6050-6062 (1999); Robinson et al, Br J Cancer 99: 1415-1425 (2008)). In addition to a high tumor uptake, a prolonged retention in the tumor is of great importance for efficient therapeutic effects of a drug. Small targeting agents, such as polypeptides derived from the Z domain, have the ability to accumulate at high levels in tumors due to high vascular permeability and rapid diffusivity into the tumor (Schmidt and Wittrup, Mol Cancer Ther 8: 2861-2871 (2009)). However, unbound proteins of low molecular weight will be cleared rapidly from the tumor and then from the circulation via the kidneys. As a consequence, a high affinity of a small-size targeting agent towards the cancer cell is of great importance for increased tumor retention. Furthermore, for efficient targeting of a receptor protein with low expression (104 target proteins, or less, per cell), recent results suggest that the affinity of small polypeptides (for example polypeptides derived from the Z domain) should be as high as possible, preferably with a binding constant, KD, in the low picomolar range or less (Tolmachev et al (2012), J Nucl Med 53(6):953-60).
Since HER3 may be expressed on the same tumor cell as other members of the EGF family, production of bispecific molecules targeting HER3 and another member of the EGF family has recently attracted some interest. Such bispecific molecules could for example be utilized as targeting vehicles for increasing the specificity of targeting in molecular imaging applications and simultaneously targeting HER3 and another antigen expressed on tumors.